Simplified platform for mosaic in vivo analysis of cellular maturation in the developing heart

Abstract

Cardiac cells develop within an elaborate electro-mechanical syncytium that continuously generates and reacts to biophysical force. The complexity of the cellular interactions, hemodynamic stresses, and electrical circuitry within the forming heart present significant challenges for mechanistic research into the cellular dynamics of cardiomyocyte maturation. Simply stated, it is prohibitively difficult to replicate the native electro-mechanical cardiac microenvironment in tissue culture systems favorable to high-resolution cellular/subcellular analysis, and current transgenic models of higher vertebrate heart development are limited in their ability to manipulate and assay the behavior of individual cells. As such, cardiac research currently lacks a simple experimental platform for real-time evaluation of cellular function under conditions that replicate native development. Here we report the design and validation of a rapid, low-cost system for stable in vivo somatic transgenesis that allows for individual cells to be genetically manipulated, tracked, and examined at subcellular resolution within the forming four-chambered heart. This experimental platform has several advantages over current technologies, chief among these being that mosaic cellular perturbations can be conducted without globally altering cardiac function. Consequently, direct analysis of cellular behavior can be interrogated in the absence of the organ level adaptions that often confound data interpretation in germline transgenic model organisms.

Adherens junction engagement regulates functional patterning of the cardiac pacemaker cell lineage

“…Consistent with previous work , our data indicated that CPCs were present in the right inflow myocardium from embryonic day 3 (E3) through E9 of avian cardiac morphogenesis (Figure 11A-D). To evaluate regional patterning of cell size, we generated mosaic hearts using in vivo transfection of a transposable expression construct encoding a palmitoylated membrane RFP reporter and GFP nuclear reporter (Figure 12A) (Goudy, Henley et al 2019) and threedimensional renderings of individual myocytes were generated to quantify cellular geometry (Figures 12B-12E). At E3, no statistical differences in cell volume or surface area were noted between CPCs and atrial WM (Figures 11E-11G).…”

“…To test whether diminished AJ formation alone could generate myocytes with CPC-like structural characteristics, we blocked AJ formation in the WM. We developed two independent shRNAs against N-Cad (Goudy, Henley et al 2019) and generated a mutated N-Cad expression construct lacking the extracellular domain required for intercellular binding (N-cad∆112-624) . When transfected into primary embryonic WM, both shRNAs and the N-cad∆112-624 construct diminished cell-cell contacts and AJ-formation in cultured primary embryonic WM (Figure 16).…”

“…Transfections were carried out as previously described (Goudy, Henley et al 2019). In brief, with 0.1% Tween-20 for 20 minutes.…”

“…In short, embryonic avian hearts will be transfected during early looping stages with integrating DNA constructs encoding either membrane targeted TagRFP and a nuclear target GFP (as controls), a Ncadherin expressing construct (polycistronic membrane TagRFP-2a-N-Cad) or an N-Cadherin blocking construct (polycistronic membrane TagRFP-2a-N-CadD116-624) (Goudy et al, 2019). In addition, to the N-cadherin overexpressing or blocking construct hearts will be transfection with a Megakaryoblastic leukemia protein-1 (MKL1) expressing construct fused to monomeric GFP ( MKL1-GFP).…”

“…Hearts will then be isolated and CPC myocardium, atrial myocardium, and ventricular myocardium will be mechanically dissected. Cells from each region will then be enzymatically dissociated and transfected cells will be collected using Fluorescence Activated Cell Sorting (FACS) according to our previously published protocols (Goudy et al, 2019). As the polycistronic construct is based on the viral-2a system, fluorescence intensity can be used to normalize the level of N-Cadherin or N-Cadherin D116-624 expression per cell.…”